Vaccines comprising TB10.4

ABSTRACT

Vaccination with the combination of Ag85B-TB10.4 and IC31® adjuvant generated a high amount of polyfunctional CD4 + T cells expressing high levels of IFN-γ, TNF-α, and IL-2. This in turn led to significant protection against infection with M. tuberculosis in the mouse aerosol challenge model of tuberculosis. Both the immunogenicity of the vaccine and its ability to protect against TB infection was highly dependent on the antigen dose. Thus, whereas the standard antigen dose of 5 μg, as well as 15 μg, did not induce significant protection against M. tuberculosis, reducing the dose to 0.5 μg increased both the immunogenicity of the vaccine as well as its protective efficacy to a level comparable to that observed in BCG vaccinated mice. Thus, the IC31® adjuvant, with the specified antigen dose, can induce a strong protective Th1 response against M. tuberculosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 12/500,881, filed Jul. 10, 2009, which claims the benefit under 35 USC 119(e) of prior U.S. Provisional Patent Application No. 61/085,973, filed Aug. 4, 2008, which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The global effort to develop a more effective Mycobacterium tuberculosis (M. tuberculosis) vaccine than the currently used Bacillus of Calmette and Guerin (BCG) vaccine involves different strategies such as live attenuated vaccines (Horwitz, et al., 2000), virally vectored M. tuberculosis vaccines (McShane, et al., 2004), and subunit vaccines (Olsen, et al., 2001 and Skeiky, et al., 2004). The subunit approach holds a number of advantages, such as increased safety and stability as well as the demonstrated ability to boost prior BCG vaccination (Brandt, et al., 2004; Dietrich, et al., 2006). In addition, as subunit vaccines appear not to be influenced by environmental mycobacteria, this type of vaccine may be of particular use in the developing world (Brandt, et al., 2002). However, progress in this field has been delayed by the lack of adjuvants that induce a strong cell-mediated immune (CMI) response. Therefore, a need still remains for an immunogenic composition which can generate polyfunctional immune cells thereby providing greater protection against M. tuberculosis.

SUMMARY OF THE INVENTION

An immunogenic composition and vaccine for mammalian use with a low dose of an antigen comprising TB10.4 fused to a polypeptide of the antigen 85-complex (Ag85, composed of the Ag85A, Ag85B, and Ag85C proteins (Dietrich, et al., 2005)), e.g., Ag85B in an adjuvant, and methods of immunization and treatment of M. tuberculosis, are provided.

An immunogenic composition for mammalian use is provided comprising a TB10.4 protein and an Ag85-complex protein (described herein), which optionally can be fused together or provided as separate proteins, wherein the total amount of protein is less than about 25 μg, or less than 10 μg or equal to about 0.5 μg per antigen dose. In a further embodiment, the composition is for human use. In one embodiment, the composition does not contain dimethyl dioctadecyl ammonium bromide (DDA). An immunogenic composition described herein can additionally comprise an adjuvant. In one embodiment, the adjuvant has at least one polycationic peptide and at least one oligonucleotide, and in a further embodiment the oligonucleotide is a TLR9 (toll-like receptor 9) agonist. In one embodiment, the adjuvant is IC31® adjuvant (described herein). In a further embodiment, the protein is from the Ag85-complex is an Ag85B protein.

In another embodiment, a vaccine is provided for mammalian use which comprises the above mentioned immunogenic composition. In a further embodiment, the vaccine is for human use. In still another embodiment, a method of inducing protection against M. tuberculosis in a mammal is provided, the method comprising introducing into the mammal an immunogenic composition as described above. In a further embodiment, the method is for inducing protection in a human.

Still other aspects and embodiments of the invention will be apparent from the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate the concentration of IFN-γreleased upon stimulation of peripheral blood mononuclear cells (PBMCs) with either 1 or 5 μg/ml of Ag85B, TB10.4, control antigen CFP10, or no antigen. Stimulation is post-vaccination with varying doses of Ag85B-TB10.4 (Hyvac4, i.e., H4) formulated in IC31® adjuvant −15 μg H4 (FIG. 1C), 5 μg H4 (FIG. 1B), 0.5 μg H4 (FIG. 1D), or control (FIG. 1A). Values represent the means of triplicate and SEMs are indicated by bars.

FIGS. 2A-2D illustrate IFN-γ released upon stimulation of PBMC's (FIG. 2A)/(FIG. 2C) and splenocytes (FIG. 2B)/(FIG. 2D) with Ag85B (FIG. 2A)/(FIG. 2B) or TB10.4 (FIG. 2C)/(FIG. 2D), post-vaccination with 0.5 μg, 5 μg, or 15 μg or 0 μg (non-vacc.) of H4 formulated in IC31® adjuvant. In (FIG. 2A)-(FIG. 2D), a vaccination dose of 0.5 μg H4 gave significantly (p<0.05, one-way ANOVA and Tukey's post test) higher antigen responses, compared to vaccination doses of 5 μs and 15 μg.

FIGS. 3A and 3B illustrate the protective efficacy of H4. FIGS. 3A and 3B reflect data obtained from two experiments (repeated). The groups of mice reflected are non-vaccinated (negative control), Bacillus of Calmette and Guerin (BCG) immunized (positive control), 0.5 μg H4 (Ag85B-TB10.4 fusion protein in IC31® adjuvant), 5 μg H4, and 15 μg H4. Following challenge with aerosolized virulent M. tuberculosis (Erdman strain), colony forming units (CFU) in the lungs were determined. Log₁₀ CFU is reflected for each group.

DETAILED DESCRIPTION OF THE INVENTION

The combination of Ag85B-TB10.4 (Hyvac 4) and the IC31® adjuvant as an immunogenic composition and as a new vaccine against infection of mammals with M. tuberculosis is provided. In a further embodiment, an immunogenic composition or vaccine as described herein is effective against infection in humans. Ag85B-TB10.4 and the IC31® adjuvant induces high amounts of polyfunctional CD4⁺ T cells and provides significant protection against M. tuberculosis. Surprisingly, the combination of the Ag85B-TB10.4 antigen and the IC31® adjuvant was sensitive to the antigen dose. Thus, whereas a standard dose in mice of 5 μg of Ag85B-TB10.4 in IC31® adjuvant did not lead to protection against M. tuberculosis, 0.5 μg Ag85B-TB10.4 in IC31® adjuvant induced protection comparable to that of Bacillus of Calmette and Guerin (BCG).

In an effort to generate an efficient vaccine against infection of mammals with M. tuberculosis, the combination of the Ag85B-TB10.4 fusion protein and IC31® adjuvant is used in one or more embodiments. Ag85B-TB10.4 fusion protein has the advantage that it does not include any of the proteins that are useful for diagnostic purposes such as, e.g., ESAT-6. The absence of ESAT-6 in a vaccine as described herein will allow diagnostic tests and a vaccine to be used in parallel since the Ag85B-TB10.4 fusion protein does not compromise any of the specific diagnostic tests. In one embodiment, a vaccine described herein contains an Ag85 protein and TB10.4 as the sole antigens. In a further embodiment, a vaccine described herein contains the fusion of Ag85B-TB10.4 as the sole antigen.

In another embodiment, the vaccine excludes (does not contain) dimethyl dioctadecyl ammonium bromide (DDA). In yet another embodiment, the vaccine excludes (does not contain) monophosphoryl lipid A (MPL). In another embodiment, the vaccine excludes (does not contain) DDA or MPL. In another embodiment, the vaccine contains a mixture a polycationic peptide and oligodeoxynucleic molecules. In a further embodiment, the vaccine contains a mixture of peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a; polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃) as the sole adjuvant. In a further embodiment, the vaccine contains IC31® adjuvant as the sole adjuvant.

The Ag85B-TB10.4 fusion protein in the IC31® adjuvant constitutes an effective vaccine against infection in mammals with M. tuberculosis. In a further embodiment, the vaccine is effective against infection in humans.

In one embodiment, a vaccine as described is useful as a BCG booster vaccine.

In another embodiment, the Ag85B-TB10.4 fusion protein in the IC31® adjuvant constitutes an effective vaccine against infection with M. tuberculosis. The Ag85B-TB10.4 and IC318 combination induces a high amount of polyfunctional CD4⁺ T cells and provides significant protection against M. tuberculosis. Surprisingly, the combination of the Ag85B-TB10.4 fusion protein and the IC31® adjuvant is extremely sensitive to the antigen dose. Whereas a dose of 5 μg of Ag85B-TB10.4 in IC31® adjuvant does not lead to significant protection against M. tuberculosis, 0.5 μg Ag85B-TB10.4 in IC31® adjuvant induces a strong protection comparable to that of BCG (Ex. 3). Applicants have identified that Ag85B-TB10.4 is an extraordinarily immunogenic molecule.

In one embodiment, the application is directed to the combination of an Ag85B-TB10.4 (Hyvac 4; H4) fusion protein and IC31® adjuvant as a new vaccine against infection with M. tuberculosis. The IC31® adjuvant comprises cationic peptides and is a TLR9 (toll-like receptor 9) agonist.

A vaccine for mammalian use with a low dose of an antigen comprising TB10.4 fused to a polypeptide of the antigen 85-complex, e.g., Ag85B in an adjuvant, and methods of immunization against, and treatment of, M. tuberculosis, are provided. In a further embodiment, the vaccine is for human use.

An immunogenic composition for mammalian use is provided comprising a TB10.4 protein and an Ag85-complex protein which optionally can be fused together or provided as separate proteins wherein the total amount of protein is less than about 25 μg, or less than 10 μg or equal to about 0.5 μg per antigen dose. In a further embodiment, the immunogenic composition is for human use. In still another embodiment, the composition excludes (does not contain) ESAT-6. In one embodiment, a composition described herein contains an Ag85 protein and TB10.4 as the sole antigens. In a further embodiment, a composition described herein contains the fusion of Ag85B-TB10.4 as the sole antigen.

In a further embodiment, the composition excludes (does not contain) dimethyl dioctadecyl ammonium bromide (DDA). In another embodiment, the composition excludes (does not contain) monophosphoryl lipid A (MPL). In another embodiment, the composition excludes (does not contain) DDA or MPL. In another embodiment, the composition contains a mixture a polycationic peptide and oligodeoxynucleic molecules. In a further embodiment, the composition contains a mixture of peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a; polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃) as the sole adjuvant. In a further embodiment, the composition contains IC31® adjuvant as the sole adjuvant.

An immunogenic composition described herein can additionally comprise an adjuvant. In one embodiment, the adjuvant has at least one polycationic peptide and at least one oligonucleotide, and in a further embodiment the oligonucleotide is a TLR9 agonist.

In one embodiment, the adjuvant is IC31®. In a further embodiment, the protein from the Ag85-complex is an Ag85B protein.

Also described is a vaccine for mammalian use comprising an immunogenic composition described herein. In a further embodiment, the vaccine is for human use.

In another embodiment, a method of inducing protection against M. tuberculosis in a mammal is provided, the method comprising introducing into the mammal an immunogenic composition as described herein. In another embodiment, a method of inducing polyfunctional CD4⁺ T cells in a mammal is provided, the method comprising introducing into the mammal an immunogenic composition as described herein. In another embodiment, a method of inducing an immune response against M. tuberculosis in a mammal is provided, the method comprising introducing into the mammal an immunogenic composition as described above. In further embodiments of these methods, the mammal is a human.

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations thereof such as “comprises” or “comprising”, will be understood to mean the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Unless otherwise indicated, the term “about” means±10% the limit of weight measurement given.

A. Antigens

In one embodiment, the antigen may comprise a protein of the Ag85-complex fused to TB10.4 protein (TB10.4 described in Dietrich, et al., 2005), including, for example, proteins Ag85A, Ag85B or Ag85C of the Ag85 complex. The proteins of the antigen 85 complex (85A, 85B, and 85C) are encoded by three genes located at different sites in the mycobacterial genome, which show extensive cross-reactivity as well as homology at amino acid and gene levels. The proteins differ slightly in molecular mass in the 30- to 31-kDa region. The individual components of the Ag85 complex (Ag85A, Ag85B and Ag85C) are publicly available (e.g., from Colorado State University).

In one embodiment, a fusion protein of the TB10.4 protein and an Ag85-complex protein may be prepared as described in [Dietrich, et al., 2005]. In another embodiment, a fusion may be prepared by linking the TB10.4 protein to an Ag85-complex protein directly or via a connecting linker of at least one amino acid. Other methods of preparing the fusion proteins are known conventionally, and are considered to be useful herein.

In another embodiment, Ag85B-TB10.4 is utilized. In a further embodiment, Ag85B-TB10.4 is given in low doses, i.e., doses less than those currently used in subunit M. tuberculosis vaccines (e.g., 5 μg to 25 μg per dose), to initiate the maximum amount of polyfunctional immune cells, inducing more interferon-γ expression and increased protection against M. tuberculosis. Throughout this specification Ag85B-TB10.4 fusion protein is interchangeable with the terms H4 and HyVac4.

In another embodiment the antigen may comprise a protein of the Ag85 complex and TB10.4 protein wherein the Ag85 complex protein is not fused to the TB10.4 protein. In a further embodiment, the protein of the Ag85 complex is Ag85B. In one embodiment, the Ag85B-TB10.4 fusion protein is prepared according to Dietrich, et al., 2005.

In still another embodiment, the antigen excludes (does not contain) ESAT-6.

Protein amino acid sequence Ag85A (SEQ MQLVDRVRGA VTGMSRRLVV GAVGAALVSG ID NO: 3) LVGAVGGTAT AGAFSRPGLP VEYLQVPSPS MGRDIKVQFQ SGGANSPALY LLDGLRAQDD FSGWDINTPA FEWYDQSGLS VVMPVGGQSS FYSDWYQPAC GKAGCQTYKW ETFLTSELPG WLQANRHVKP TGSAVVGLSM AASSALTLAI YHPQQFVYAG AMSGLLDPSQ AMGPTLIGLA MGDAGGYKAS DMWGPKEDPA WQRNDPLLNV GKLIANNTRV WVYCGNGKPS DLGGNNLPAK FLEGFVRTSN IKFQDAYNAG GGHNGVFDFP DSGTHSWEYW GAQLNAMKPD LQRALGATPN TGPAPQGA Ag85B (SEQ MTDVSRKIRA WGRRLMIGTA AAVVLPGLVG ID NO: 4) LAGGAATAGA FSRPGLPVEY LQVPSPSMGR DIKVQFQSGG NNSPAVYLLD GLRAQDDYNG WDINTPAFEW YYQSGLSIVM PVGGQSSFYS DWYSPACGKA GCQTYKWETF LTSELPQWLS ANRAVKPTGS AAIGLSMAGS SAMILAAYHP QQFIYAGSLS ALLDPSQGMG PSLIGLAMGD AGGYKAADMW GPSSDPAWER NDPTQQIPKL VANNTRLWVY CGNGTPNELG GANIPAEFLE NFVRSSNLKF QDAYNAAGGH NAVFNFPPNG THSWEYWGAQ LNAMKGDLQS SLGAG Ag85C (SEQ MTFFEQVARL RSAATTLPRR LAIAAMGAVL ID NO: 5) VYGLVGTFGG PATAGAFSRP GLPVEYLQVP SASMGRDIKV QFQGGGPHAV YLLDGLRAQD DYNGWDINTP AFEEYYQSGL SVIMPVGGQS SFYTDWYQPS QSNGQNYTYK WETFLTREMP AWLQANKGVS PTGNAAVGLS MSGGSALILA AYYPQQFPYA ASLSGFLNPS EGWWPTLIGL AMNDSGGYNA NSMWGPSSDP AWKRNDPMVQ IPRLVANNTR IWVYCGNGTP SDLGGDNIPA KFLEGLTLRT NQTFRDTYAA DGGRNGVFNF PPNGTHSWPY WNEQLVAMKA DIQHVLNGAT PPAAPAAPAA TB10.4 (SEQ MSQIMYNYPA MLGHAGDMAG YAGTLQSLGA ID NO: 6) EIAVEQAALQ SAWQGDTGIT YQAWQAQWNQ AMEDLVRAYH AMSSTHEANT MAMMARDTAE AAKWGG

Each protein may be modified by glycosylation, or lipidation (Mowat, et al., 1991; Lustig, et al., 1976). Each protein may be modified by the addition of prosthetic groups, a purification moiety, or a signal peptide. Each protein may be modified one or more times or not undergo any modification. Each protein may be modified singly or in combination. Each protein will be characterised by specific amino acids and be encoded by specific nucleic acid sequences. Within the scope of the present invention are such sequence and analogues and variants produced by recombinant or synthetic methods wherein such amino acid sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant protein while retaining immunogenicity as confirmed by any one or all of the biological assays described herein.

Substitutions are preferably “conservative”. These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code.

ALIPHATIC Non-polar G, A, P I, L, V Polar-uncharged C, S, T, M N, Q Polar-charged D, E K, R AROMATIC H, F, W,Y B. Adjuvants

In one embodiment, the antigen comprises an Ag85 complex protein fused to TB10.4 protein in an adjuvant. In another embodiment, the antigen comprises an Ag85 complex protein and a TB10.4 protein (i.e., non-fused) in an adjuvant.

Ag85B-TB10.4 is an extraordinary immunogenic molecule which must be given in low doses to initiate the maximum amount of polyfunctional immune cells inducing more interferon-γ expression and increased protection against M. tuberculosis. It is exemplified with an adjuvant comprising a polycationic peptide [e.g., polylysine (KLK peptide) or polyarginine] and oligodeoxynucleic molecules but is not limited to this adjuvant.

In a further embodiment, the adjuvant is a mixture of a polycationic peptide [e.g., polylysine (KLK peptide) or polyarginine] and oligodeoxynucleic molecules [I-ODNs]. An example of a suitable I-ODN for use with the current invention is oligo dIC, e.g., oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a; polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃). I-ODN's appropriate for use in the embodiments described herein may be found in International (PCT) Patent Application Publication Nos.: WO 01/93905 and WO 01/93903, which are hereby incorporated by reference. An example of KLK peptides suitable for use with the current invention are NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) or KLKLLLLLKLK-NH₂ (SEQ ID NO:1). Additional examples of appropriate polycationic peptides are RLRLLLLLRLR-NH₂ (SEQ ID NO:7), RLKLLLLLKLR-NH₂ (SEQ ID NO:8), KFKFFFFFKFK-NH₂ (SEQ ID NO:9), KWKWWWWWKWK-NH₂ (SEQ ID NO:10) or KVKVVVVVKVK-NH₂ (SEQ ID NO:11). Polycationic peptides suitable for use in these embodiments may be found in International (PCT) Patent Application Publication No. WO/0232451, which is hereby incorporated by reference.

As described herein, the IC31® adjuvant (Intercell AG) comprises a mixture of the peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) (Multiple Peptide Systems, San Diego, Calif., USA) and the oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a; polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃) (Proligo, Boulder, USA).

In one embodiment, a dose of 0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant [100 nmol peptide (NH₂-KLKLULLKLK-COOH (SEQ ID NO:1) and 5 nmol oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃] induces a strong INF-γ response. However, one of skill in the art will be able to adjust amounts and concentrations according to the application. This strong IFN-γ response is in approximately the same range as 5 μg Ag85B-TB10.4 fusion protein, without IC31® adjuvant. In a further embodiment, 0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant [100 nmol peptide (NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and 5 nmol oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃] is utilized. This provides a strong protection in approximately the same range as BCG. Without wishing to be bound by theory, Applicants have determined that this is surprisingly better than 5 μg or 15 μg of Ag85B-TB10.4 fusion protein alone (i.e., no IC31® adjuvant), revealing that the preferred dose of Ag85B-TB10.4 fusion protein alone may be lower.

In one embodiment, less than 5 μg of Ag85B-TB10.4 fusion protein per dose is utilized. In still other embodiments, 1 μg, 0.5 μg, or 0.1 μg of Ag85B-TB10.4 fusion protein is utilized.

As reflected herein, the lowest dose of Ag85B-TB10.4 fusion protein (0.5 μg) in IC31® adjuvant gave the highest IFN-γ response after stimulation with either of the vaccine components (see FIGS. 1A-1D and 2A-2D). Finally, vaccination with Ag85B-TB10.4 fusion protein in IC31® adjuvant induced two major CD4⁺ T cell populations, one expressing IFN-γ, IL-2, and TNF-α, and another expressing IL-2 and TNF-α. Both of these T cell populations belong to central memory T cells, and are necessary for long term memory. Importantly, as seen with the IFN-γ expression measured by ELISA, using a dose of 0.5 μg HyVac4 fusion protein in IC31® adjuvant induced the highest cell numbers within the polyfunctional population that expressed IFN-γ, IL-2, and TNF-α.

Applicants have determined that the adjuvants IC31® and cationic liposomes exhibit different sensitivities towards the antigen dose; and that the preferred antigen dose in IC31® adjuvant is antigen-dependent.

0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant induced significant protection whereas 5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant did not (FIGS. 3A-3B). One of skill in the art will recognize from this application that the appropriate antigen dose in a vaccine depends both on the antigen and on the adjuvant.

In one embodiment, the composition excludes (does not contain) dimethyl dioctadecyl ammonium bromide (DDA). In another embodiment, the composition excludes (does not contain) monophosphoryl lipid A (MPL).

The following examples are illustrative of the compositions and methods of the invention. It will be readily understood by one of skill in the art that the specific conditions described herein can be varied without departing from the scope of the present invention. It will be further understood that other compositions not specifically illustrated are within the scope of the invention as defined herein.

EXAMPLES

The following information is supportive of the examples that follow.

Animals:

Studies were performed with 8 to 12 week-old C57BL/6xBalb/c F1 female mice, purchased from Taconic, Ejby, Denmark. Infected animals were housed in cages contained within laminar flow safety enclosures in a BSL-3 facility. The use of mice was in accordance with the regulations set forward by the Danish Ministry of Justice and Animal Protection Committees and in compliance with EC Directive 86/609 and the US ALAC recommendations for the care and use of Laboratory animals.

Bacteria:

M. tuberculosis Erdman were grown at 37° C. on Löwenstein-Jensen medium or in suspension in Sauton medium enriched with 0.5% sodium pyruvate and 0.5% glucose.

Immunization:

Mice were immunized three times at 2-week intervals subcutaneously on the back with experimental vaccines containing 0.5, 5 or 15 μg of Ag85B-TB10.4 fusion protein (H4)/dose, emulsified in IC31® adjuvant in a total volume of 0.2 ml/dose. Doses were 100 nmol peptide and 5 nmol oligonucleotide. All vaccines were formulated using 10 mM Tris-HCl/270 mM sorbitol buffer (pH 7.9) as previously described (Olsen, et al., 2001) to obtain a final volume of 0.2 ml/mouse. At the time of the first subunit vaccination, one group of mice received a single dose of BCG Danish 1331 (2.5×10⁵ CFU) injected subcutaneously at the base of the tail and one group received a saline injection. All groups of mice were challenged 10 weeks after the first vaccination.

Experimental Infections:

When challenged by the aerosol route, the animals were infected with approximately 50 CFU of M. tuberculosis Erdman/mouse. These mice were sacrificed 6 weeks after challenge. Numbers of bacteria in the spleen or lung were determined by serial threefold dilutions of individual whole-organ homogenates in duplicate on 7H11 medium (Middlebrook; Sigma-Aldrich). Organs from the BCG-vaccinated animals were grown on medium supplemented with 2 μg of 2-thiophene-carboxylic acid hydrazide (TCH)/ml to selectively inhibit the growth of the residual BCG bacteria in the test organs. Colonies were counted after 2 to 3 weeks of incubation at 37° C. Bacterial burden in the lungs was expressed as log₁₀ of the bacterial counts based on vaccination groups of six animals.

Lymphocyte Cultures:

Lymphocytes from spleens were obtained as described previously (Brandt, et al.). Blood lymphocytes (PBMCs) were purified on a density gradient. Cells pooled from five mice in each experiment were cultured in microtiter wells (96-well plates; Nunc, Roskilde, Denmark) containing 2×10⁵ cells in a volume of 2000 of RPMI 1640 supplemented with 5×10⁻⁵ M 2-mercaptoethanol, 1% penicillin-streptomycin, 1 mM glutamine, and 5% (vol/vol) fetal calf serum. Based on previous dose-response investigations, the mycobacterial antigens were all used at 15 μg/ml or 5 μg/ml, while concanavalin A was used at a concentration of 1 μg/ml as a positive control for cell viability. All preparations were tested in cell cultures and found to be nontoxic at the concentrations used in the present study. Supernatants were harvested from cultures after 72 h of incubation for the investigation of IFN-γ.

IFN-γ Enzyme-Linked Immunosorbent Assay (ELISA):

Microtiter plates (96 wells; Maxisorb; Nunc) were coated with monoclonal hamster anti-murine IFN-γ (Genzyme, Cambridge, Mass.) in PBS at 4° C. Free binding sites were blocked with 1% (wt/vol) bovine serum albumin-0.05% Tween 20. Culture supernatants were tested in triplicate, and IFN-γ was detected with a biotin-labelled rat anti-murine monoclonal antibody (clone XMG1.2; Pharmingen, San Diego, Calif.). Recombinant IFN-γ (Pharmingen, San Diego, Calif.) was used as a standard.

FACS Analysis of Lymphocytes:

Intracellular cytokine staining procedure: Cells from blood, spleen or lungs of mice were stimulated for 1-2 h with 2 μg/ml Ag and subsequently incubated for 6 h with 10 μg/ml brefeldin A (Sigma-Aldrich, USA) at 37° C. Thereafter, cells were stored overnight at 4° C. The following day, Fc receptors were blocked with 0.54 ml anti-CD16/CD32 mAb (BD Pharmingen, USA) for 10 minutes, where after the cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS), and stained for surface markers as indicated using 0.2 μg/ml anti-CD4 (clone: RM4-5), anti-CD8 (clone: 53-6,7) mAb's. Cells were then washed in FACS buffer, permeabilized using the Cytofix/Cytoperm™ kit (BD Pharmingen, Denmark) according to the manufacturers instructions, and stained intracellularly with 0.2 μg/ml anti-IFN-γ (clone: XMG1.2), anti-TNF-α (clone: MP6-XT22), or anti-IL-2 (clone: JES6-5H4) mAb's. After washing, cells were re-suspended in formaldehyde solution 4% (w/v) pH 7.0 (Bie & Berntsen, Denmark) and analysed by flow cytometry on a six-colour BD FACSCanto flow cytometer (BD Biosciences, USA).

Statistical Methods:

The data obtained were tested by analysis of variance. Differences between means were assessed for statistical significance by Tukey's test. A P value of <0.05 was considered significant.

Example 1 Immune Response Induced after Immunization with Ag85B-TB10.4 Fusion Protein in IC31® Adjuvant

The immunogenicity of Ag85B-TB10.4 fusion protein delivered in IC31® adjuvant was analyzed, including whether both components of the fusion protein were recognized by the immune system after immunization.

PBMC's isolated from groups of mice vaccinated with different doses of H4 in IC31® adjuvant and a saline control group were stimulated with either 1 or 5 μg/ml of Ag85B, TB10.4 or CFP10 (a Mycobacterium tuberculosis-specific antigen). After 72 hours the concentration of cell released IFN-γ was determined by ELISA. PBMC's were isolated 1 week after third vaccination and were pooled from five mice per group. Values in FIGS. 1A-1D represent the means of triplicate and SEM's are indicated by bars.

Groups of mice were immunized with Ag85B-TB10.4 fusion protein in IC31® adjuvant. As negative control, a group of mice received the adjuvant alone (data not shown). To examine antigen dose in IC31® adjuvant, we used 15, 5 and 0.5 μg of Ag85B-TB10.4 fusion protein. One week after the last injection, mice were bled, and the IFN-γ release was evaluated after in vitro stimulation of purified PBMCs with different concentrations of Ag85B and TB10.4 proteins (5 μg/ml and 1 μg/ml) (FIG. 1A). Immunization with Ag85B-TB10.4 fusion protein in IC31® adjuvant induced a strong IFN-γ response specific for Ag85B and TB10.4 proteins (FIGS. 1B-1D). Surprisingly, this response was sensitive to the antigen immunization dose. Thus, the lowest dose of Ag85B-TB10.4 fusion protein in IC31® adjuvant gave the highest IFN-γ response after stimulation with either Ag85B (9401+/−3668 pg/ml IFN-γ) or TB10.4 (4694+/−3992 pg/ml IFN-γ) (FIG. 1D). Using a dose of 5 μg or 15 μg Ag85B-TB10.4 fusion protein significantly reduced the IFN-γresponse against both Ag85B and TB10.4 proteins relative to mice vaccinated with 0.5 μg Ag85B-TB10.4 fusion protein (p<0.001). This was particularly apparent for the high immunization dose −15 μg Ag85B-TB10.4 fusion protein per immunization dose (FIG. 1C)—which gave IFN-γ responses that did not differ from the observed responses in non-vaccinated mice (or in Ag85B-TB10.4 fusion protein vaccinated mice stimulated in vitro with control antigen CFP10).

The same dose dependency was subsequently repeated in an independent experiment where the immune responses were analyzed in both blood and spleen (FIGS. 2A-2D). PBMC's (2A)/(2C) and splenocytes (2B)/(2D) isolated from groups of mice immunized with 3 different doses of H4 formulated in IC31® adjuvant or a saline control group were stimulated with Ag85B (2A)/(2B) or TB10.4 (2C)/(2D) for 72 hours where after IFN-γ cytokine secretion was measured by ELISA. The bars represent means of 3 individual mice. SEMs are indicated. In FIGS. 2A-2D, a vaccination dose of 0.5 μg H4 gave significantly (p<0.05, one-way ANOVA and Tukey's post test) higher antigen responses, compared to vaccination doses of 5 μg and 15 μg.

These results show that the lowest dose of 0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant induced the strongest systemic response of the antigen doses tested.

Example 2 Vaccination with Ag85B-TB10.4 Fusion Protein in IC31® Adjuvant Induces Polyfunctional CD4⁺T Cells

The phenotype of the T cells induced by immunizing with Ag85B-TB10.4 fusion protein in IC31® adjuvant was analyzed. In particular, the ability of this vaccine to induce polyfunctional (IFN-γ⁺IL-2⁺TNF-α⁺) CD4⁺ T cells was determined as these have been shown to correlate with protective immunity against infections such as Leishmania major and to faun the basis for a long lived memory response.

Ag85B and TB10.4 specific T cells are poly-functional. Production of IFN-γ, TNF-α and IL-2 was assessed following antigenic stimulation of PBMC's and spleenocytes 2 weeks post-vaccination by flow cytometry.

PBMC's from Ag85B-TB10.4 fusion protein in IC31® adjuvant vaccinated mice were stimulated in vitro with Ag85B or TB10.4 fusion protein and analyzed by flow cytometry for expression of CD4, CD8, TNF-γ, TNF-α, and IL-2. The results show that immunizing with Ag85B-TB10.4 fusion protein in IC31® adjuvant induced two major polyfunctional T cell populations; CD4⁺IFN-γ⁺IL-2⁺TNF-α⁺ and CD4⁺IL-2⁺TNF-α⁺ T cells. This was seen for Ag85B and TB10.4 specific T cells. Interestingly, as observed in FIG. 1, there is a higher response in the group immunized with 0.5 μg HyVac4 in IC31® adjuvant compared to the group immunized with 5 μg HyVac4 fusion protein in IC31® adjuvant, and that the major difference was that the group being vaccinated with only 0.5 μg showed significantly more polyfunctional T cells. Taken together, immunizing with Ag85B-TB10.4 fusion protein in IC31® adjuvant induced polyfunctional CD4⁺ T cells and confirmed that lowering the amount of Ag85B-TB10.4 fusion protein increased the immunogenicity of the vaccine in terms of not only IFN-γexpression but also the number of polyfunctional T cells.

Example 3 Protective Efficacy of Ag85B-TB10.4 Fusion Protein and IC31® Adjuvant in a Mouse M. Tuberculosis Infection Model

The protective efficacy of Ag85B-TB10.4 fusion protein in IC31® adjuvant was examined, including whether the dose dependency regarding the immunogenicity of the vaccine was also reflected in the protective efficacy of the vaccine.

In two independent experiments (A and B) groups of mice were vaccinated with three different doses of H4 formulated in IC31® adjuvant and compared to saline and BCG-vaccinated controls.

Mice were vaccinated three times at two weeks interval with Ag85B-TB10.4 fusion protein in IC31® adjuvant. As a positive control for protection, a group of mice were immunized once with BCG.

Ten weeks after the first vaccination, the mice were challenged by the aerosol route with virulent M. tuberculosis Erdman. Six weeks post challenge, the mice were sacrificed and the numbers [bacterial burden (CFU)] were determined in the lungs. As observed with the immunogenicity of the vaccines, the lowest Ag85B-TB10.4 fusion protein immunization dose induced the highest protection. Thus, mice vaccinated with 0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant contained a bacterial number of 5.0+/−0.2 Log₁₀ CFU in the lungs. This was equal to the numbers observed in BCG vaccinated mice (4.90+/−0.35 Log₁₀ CFU), but significantly lower (p<0.001) compared to the bacterial numbers in non-vaccinated mice (5.83+/−0.12 Log₁₀ CFU) (FIG. 3A). In contrast, the bacterial numbers in mice vaccinated with 5 μg or 15 μg of Ag85B-TB10.4 fusion protein in IC31® adjuvant, were not significantly different from the levels found in the lungs of non-vaccinated mice (FIG. 3A). Repeating the experiment led to the same conclusion although the overall bacterial numbers were slightly lower in all the groups (FIG. 3B). Thus the ability of the vaccine, Ag85B-TB10.4 fusion protein in IC31® adjuvant, to induce protection against M. tuberculosis correlated with the immunogenicity of the vaccine, in terms of IFN-γproduction and the number of polyfunctional T cells, and was highest when the lowest antigen dose was used.

In both experiments, data are presented as mean values from six animals per group and standard errors of the means are indicated by bars. Statistical comparisons among the vaccination groups were done by one-way ANOVA and Tukey's post test. Significant differences are only shown for selected groups. ***: p<0.001, *: p<0.05.

The surprising in vivo results from these well recognized M. tuberculosis animal models, supports the use of the immunogenic compositions of the current invention as a M. tuberculosis vaccine in humans.

Example 4 Protective Efficacy of Ag85B-TB10.4 Fusion Protein and IC31® Adjuvant in a Clinical Trial

In human clinical trials subjects will be vaccinated with less than about 5 μg to 25 μg of Ag85B-TB10.4 in IC31® adjuvant. This low dose of Ag85B-TB10.4 is in stark contrast with other subunit M. tuberculosis vaccines currently in clinical trials. For example, 40 μg of MTB72F in AS02A per dose (Leroux-Roels, et al., 2005) and 50 μg of Ag85B-ESAT-6 in IC31® adjuvant per dose (clinical data not published yet).

PUBLICATIONS

-   1. Agger, E. M., I. Rosenkrands, A. W. Olsen, G. Hatch, A.     Williams, C. Kritsch, K. Lingnau, A. von Gabain, C. S.     Andersen, K. S. Korsholm, and P. Andersen. 2006. Protective immunity     to tuberculosis with Ag85B-ESAT-6 in a synthetic cationic adjuvant     system IC31. Vaccine 24:5452-5460. -   2. Brandt, L., M. Elhay, I. Rosenkrands, E. B. Lindblad, and P.     Andersen. 2000. ESAT-6 subunit vaccination against Mycobacterium     tuberculosis. Infect Immun 68:791-795. -   3. Brandt, L., J. Feino Cunha, A. Weinreich Olsen, B. Chilima, P.     Hirsch, R. Appelberg, and P. Andersen. 2002. Failure of the     Mycobacterium bovis BCG vaccine: some species of environmental     mycobacteria block multiplication of BCG and induction of protective     immunity to tuberculosis. Infect Immun 70:672-678. -   4. Brandt, L., Y. A. Skeiky, M. R. Alderson, Y. Lobet, W.     Dalemans, O. C. Turner, R. J. Basaraba, A. A. Izzo, T. M.     Lasco, P. L. Chapman, S. G. Reed, and I. M. Orme. 2004. The     protective effect of the Mycobacterium bovis BCG vaccine is     increased by coadministration with the Mycobacterium tuberculosis     72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected     guinea pigs. Infect Immun 72:6622-6632. -   5. Brock, I., K. Weldingh, E. M. Leyten, S. M. Arend, P. Ravn,     and P. Andersen. 2004. Specific T-cell epitopes for     immunoassay-based diagnosis of Mycobacterium tuberculosis infection.     J Clin Microbiol 42:2379-2387. -   6. Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. Lindsay, D. F.     Davey, B. J. Flynn, S. T. Hoff, P. Andersen, S. G. Reed, S. L.     Morris, M. Roederer, and R. A. Seder. 2007. Multi-functional TH1     cells define a correlate of vaccine-mediated protection against     Leishmania major. Nat Med 13:843-850. -   7. Dietrich, J., C. Andersen, R. Rappuoli, T. M. Doherty, C. G.     Jensen, and P. Andersen. 2006. Mucosal administration of     Ag85B-ESAT-6 protects against infection with Mycobacterium     tuberculosis and boosts prior bacillus Calmette-Guerin immunity. J     Immunol 177:6353-6360. -   8. Dietrich, J., C. Aagaard, R. Leah, A. W. Olsen, A. Stryhn, T. M.     Doherty, and P. Andersen. 2005. Exchanging ESAT6 with TB10.4 in an     Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient     protection and ESAT6-based sensitive monitoring of vaccine efficacy.     J Immunol 174:6332-6339. -   9. Horwitz, M. A., G. Harth, B. J. Dillon, and S.     Maslesa-Galic. 2000. Recombinant bacillus calmette-guerin (BCG)     vaccines expressing the Mycobacterium tuberculosis 30-kDa major     secretory protein induce greater protective immunity against     tuberculosis than conventional BCG vaccines in a highly susceptible     animal model. Proc Natl Acad Sci U S A 97:13853-13858. -   10. Lalvani, A., A. A. Pathan, H. McShane, R. J. Wilkinson, M.     Latif, C. P. Conlon, G. Pasvol, and A. V. Hill. 2001. Rapid     detection of Mycobacterium tuberculosis infection by enumeration of     antigen-specific T cells. Am J Respir Crit Care Med 163:824-828. -   11. Lustig T V, Rieger H L, Kraft S C, Hunter R, Rothberg R M. 1976,     Cell Immunol 24(1):164-7. -   12. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C.     Gilbert, K. Huygen, H. A. Fletcher, and A. V. Hill. 2004.     Recombinant modified vaccinia virus Ankara expressing antigen 85A     boosts BCG-primed and naturally acquired antimycobacterial immunity     in humans. Nat Med 10:1240-1244. -   13 Mowat A M, Donachie A M, Reid G, Jarrett 0.1991, Immunology     72(3):317-22 -   14. Olsen, A. W., L. A. van Pinxteren, L. M. Okkels, P. B.     Rasmussen, and P. Andersen. 2001. Protection of mice with a     tuberculosis subunit vaccine based on a fusion protein of antigen     85b and esat-6. Infection and Immunity 69:2773-2778. -   15. Ravn, P., A. Demissie, T. Eguale, H. Wondwosson, D. Lein, H.     Amoudy, A. S. Mustafa, A. K. Jensen, A. Holm, I. Rosenkrands, F.     Oftung, J. Olobo, C. F. von-Reyn, and P. Andersen. 1999. Human T     cell responses to the ESAT-6 antigen from Mycobacterium     tuberculosis. J. Infect. Dis. 179:637-645. -   16. Skeiky, Y. A., M. R. Alderson, P. J. Ovendale, J. A.     Guderian, L. Brandt, D. C. Dillon, A. Campos-Neto, Y. Lobet, W.     Dalemans, I. M. Orme, and S. G. Reed. 2004. Differential immune     responses and protective efficacy induced by components of a     tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or     recombinant protein. J Immunol 172:7618-7628. -   17. Stockinger, B., C. Bourgeois, and G. Kassiotis. 2006. CD4+     memory T cells: functional differentiation and homeostasis. Immunol     Rev 211:39-48. -   18. Weinreich Olsen, A., L. A. van Pinxteren, L. Meng Okkels, P.     Birk Rasmussen, and P. Andersen. 2001. Protection of mice with a     tuberculosis subunit vaccine based on a fusion protein of antigen     85b and esat-6. Infect Immun 69:2773-2778. -   19. Wu, C. Y., J. R. Kirman, M. J. Rotte, D. F. Davey, S. P.     Perfetto, E. G. Rhee, B. L. Freidag, B. J. Hill, D. C. Douek,     and R. A. Seder. 2002. Distinct lineages of T(H)1 cells have     differential capacities for memory cell generation in vivo. Nature     immunology 3:852-858. -   20. US FDA. 1995. Guidance for industry: Estimating the Maximum Safe     Starting Dose in initial clinical trials for therapeutics in adult     healthy volunteers. U.S. Department of Health and Human Services,     Food and Drug Administration, Center for Drug Evaluation and     Research. -   21. Leroux-Roels I, Leroux-Roels G, Ofori-Anyinam O et al. Safety     and immunogenicity of the Mtb72f/AS02A tuberculosis vaccine in     PPD-negative Belgian adults. Medical and Health in the Tropics.     Marseille, France, 11-15 Sep. 2005 (Abstract 0-036).

All publications listed in this specification are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims. 

The invention claimed is:
 1. A method of inducing an immune response against M. tuberculosis in a mammal human, the method comprising introducing into said mammal human an immunogenic composition comprising a TB10.4 protein, an Ag85-complex protein, and an adjuvant comprising at least one polycationic peptide and at least one polydeoxyinosinic-deoxycytidylic acid, wherein the total amount of the TB10.4 protein and the Ag85-complex protein is less than about 1 μg about 5 μg to about 25 μg.
 2. The method according to claim 1, wherein the TB10.4 protein and the Ag85-complex protein are present as a fusion protein.
 3. The method according to claim 2, wherein the fusion protein is present in an amount equal to about 0.5 μg 10 μg to about 25 μg.
 4. The method according to claim 2 3, wherein the fusion protein is present in an amount equal to about 0.1 μg 15 μg.
 5. The method according to claim 1, wherein the Ag85-complex protein is an Ag85B protein.
 6. The method according to claim 1, wherein the at least one oligonucleotide is a TLR9 agonist.
 7. The method according to claim 1, wherein the adjuvant is a mixture of [peptide] NH2-KLKLLLLLKLK-COOH (SEQ ID NO: 1) and [oligonucleotide] 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2).
 8. The method according to claim 1, wherein said mammal is a human.
 9. A method of inducing protection against M. tuberculosis in a mammal human, the method comprising introducing into said mammal human an immunogenic composition comprising a TB10.4 protein, an Ag85-complex protein, and an adjuvant comprising at least one polycationic peptide and at least oligonucleotide-5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2), wherein the total amount of the TB10.4 protein and the Ag85-complex protein is less than about 1 μg about 5 μg to about 25 μg.
 10. The method according to claim 9, wherein the TB10.4 protein and the Ag85-complex protein are present as a fusion protein.
 11. The method according to claim 10, wherein the fusion protein is present in an amount equal to about 0.5 μg 10 μg to about 25 μg.
 12. The method according to claim 10 11, wherein the fusion protein is present in an amount equal to about 0.1 μg 15 μg.
 13. The method according to claim 9, wherein the Ag85-complex protein is an Ag85B protein.
 14. The method according to claim 9, wherein the at least one oligonucleotide is a TLR9 agonist.
 15. The method according to claim 9, wherein the adjuvant is a mixture of peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2).
 16. The method according to claim 9, wherein said mammal is a human.
 17. A method of inducing an immune response against M. tuberculosis in a mammal human, the method comprising introducing into said mammal human an immunogenic composition comprising a TB10.4 protein, an Ag85-complex protein, and an adjuvant comprising at least one polycationic peptide and at least one polydeoxyinosinic-deoxycytidylic acid, wherein the total amount of the TB10.4 protein and the Ag85-complex protein ranges from 0.1 μg to 1 μg is about 15 μg.
 18. The method according to claim 17, wherein the TB10.4 protein and the Ag85-complex protein are present as a fusion protein.
 19. The method according to claim 18, wherein the fusion protein is present in an amount equal to about 0.5 μg.
 20. The method according to claim 17, wherein the adjuvant is a mixture of [peptide] NH2-KLKLLLLLKLK-COOH (SEQ ID NO: 1) and [oligonucleotide] 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2).
 21. The method according to claim 1, wherein the total amount of the TB10.4 protein and the Ag85-complex protein is about 10 μg to about 25 μg.
 22. The method according to claim 21, wherein the total amount of the TB10.4 protein and the Ag85-complex protein is about 15 μg.
 23. The method according to claim 9, wherein the total amount of the TB10.4 protein and the Ag85-complex protein is about 15 μg. 